DESCRIPTION A major question in developmental neurobiology is how neuronal cells develop into their mature phenotypes. This proposal investigates the development of the Purkinje neuron in relation to one of its afferents, cerebellar granule cells. These developmental processes are examined in a noel culture system in which purified Purkinje cells undergo full dendritic differentiation when co-cultured with purified granule cells as well as in cerebellar slices. The culture system provides a unique opportunity to study the dynamics of Purkinje cell development nd its interactions with one of its presynaptic cells without the confounding influence of many other cell types that have been present in previous work. In the first set of experiments the dynamics of behavior of developing dendritic spines of the Purkinje cells will be characterize in culture and slices using time lapse microscopy. These experiments will provide a bioassay for testing the role of agents that could be provided by granule cells in the development of dendritic spines. As a first step, the role of BDNF in spine formation will be tested. In the second set of experiments ultrastructural and molecular analysis of Purkinje-granule cell synapse formation will be performed. The process of assembly of molecular components of a functional synapse will be monitored on both the structural (electron microscopy and immunocytochemistry) and functional level (synaptic vesicle recycling. These studies will provide much needed information about the process of development of an important central synapse.